One of the key issues in biotechnology is the redesign of microorganisms to optimize the yield of products of interest, such as biofuels, bulk and fine chemicals, or molecules of medical interest. The redesign modifies the metabolic flux distribution such that, ideally, the cells switch from growth, i.e., biomass production, to product synthesis. Industrial biotechnology processes (Chotani et al. (2000) Biochim. Biophys. Acta 1543:434-455) are thus typically split into two stages: (i) while already producing the target compound, a cellular population is grown to a desired size; since most of the available energy is used for biomass formation, the product yield is small; (ii) Cellular growth is shut down to uncouple the production of the target compound from biomass formation (Sonderegger et al. (2005) Metab. Eng. 7:4-9). Growth arrest occurs spontaneously when the cell density reaches a high value, but this situation is characterized by a high morbidity and drastic reduction of metabolic activity: substrate intake fluxes decrease, which severely impairs process productivity.
Maintaining high metabolic activity in the absence of growth is a fundamental problem in biotechnological engineering, since it represents “a phenotype that does not normally exist in the natural environment and which is not straightforward to engineer genetically” (Sonderegger et al. (2005) Metab. Eng. 7:7).
Metabolic engineering has proposed several ways to achieve growth arrest while maintaining metabolic activity. Some are based on targeted genetic modifications that (in)activate specific components of the cell contributing to biomass formation, such as cell-cycle arrest by overexpression of a small RNA regulating cell division (Rowe and Summers (1999) Appl. Environ. Microbiol. 65:2710-2715) or engineering of sigma factors and other global regulatory proteins (Alper et al. (2007) Metab. Eng. 9:258-267; Santos and Stephanopoulos (2008) Curr. Opin. Chem. Biol. 12:168-176). Other strategies rely on shifting the bioreactor to starvation conditions: nitrogen starvation, for example, leads to growth arrest by stopping amino acid synthesis, while phosphate starvation prevents the production of nucleotides.
These genetic or physiological perturbations have a number of drawbacks. For instance, the modification of the expression of selected enzymes may lead to imbalances in the metabolic pathway, resulting in a metabolic burden on the cell detrimental to the production rate of the target compound (Glick (1995) Biotechnol. Adv. 13:247-261; Pitera et al. (2007) Metabol. Eng. 9:193-207). Nitrogen starvation is not possible when the target compound itself has nitrogen atoms (amino acid, polyamides . . . ), while phosphate deprivation causes perturbation on cell energetic processes detrimental to the production (Baek et al. (2007) J. Microbiol. Biotechnol. 17:244-252).
There is therefore still a need for improved biological methods of production of metabolites, peptides or recombinant proteins that do not present these drawbacks and that preferably allow increasing the yield of production of the molecule of interest.